Anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors with sub-micromolar potency in the cell-based replicon assay

Article abstract of DOI:10.1016/j.bmcl.2013.09.102

Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1 and 2 resulted in the identification of multiple structural elements that contributed to improved cell culture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic (IC₅₀) and cell culture (EC₅₀) potencies of less than 100 nanomolar.

Full text of DOI:10.1016/j.bmcl.2013.09.102

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6879–6885
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase
inhibitors with sub-micromolar potency in the cell-based
replicon assay
a
a
a
a
Timothy A. Stammers a, , René Coulombe , Martin Duplessis , Gulrez Fazal , Alexandre Gagnon ,
Michel Garneau b, Sylvie Goulet a, Araz Jakalian a, Steven LaPlante a, Jean Rancourt a,
Bounkham Thavonekham a, Dominik Wernic a, George Kukolj b, Pierre L. Beaulieu a
⇑
a Medicinal Chemistry Department, Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
b Biology Department, Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2013
Revised 27 September 2013
Accepted 30 September 2013
Available online 8 October 2013
Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1
and 2 resulted in the identification of multiple structural elements that contributed to improved cell cul-
ture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic
(IC50) and cell culture (EC50) potencies of less than 100 nanomolar.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
HCV NS5B polymerase
Thumb Pocket 2
Non-nucleoside inhibitor
Structure-based drug design
Direct-acting antiviral
Direct-acting antiviral (DAA) inhibitors of hepatitis C virus
(HCV) replication are having a profound impact on treatment par-
adigms. The recently approved HCV protease inhibitors telaprevir
and boceprevir increased sustained virologic response (SVR) rates
for genotype 1 infections to 68–75% in combination with pegylated
The isomeric phenoxy substituted anthranilic acid sulfonamides
1 and 2 were found to be selective inhibitors of the NS5B polymer-
ase. While these compounds were found to possess sub-micromo-
lar potency in enzymatic assays,12 they lacked cell culture activity
in the HCV subgenomic replicon assay.13 In this communication we
report the optimization efforts that resulted in attaining sub-
micromolar cell culture potency for the anthranilic acid class of
TP-2 inhibitors.
interferon-a (pegIFN) and ribavirin compared to 40–50% for peg-
IFN and ribavirin alone (previous standard of care).1 Despite the
improvement in efficacy, these pegIFN-based therapies remain
poorly tolerated2 and significant proportions of patients are con-
traindicated for treatment3 or are untreated for other reasons.4
DAAs cannot be used alone due to rapid emergence of resistance
but interferon-free combinations of two or three complementary
DAAs have achieved improved SVRs and will provide HCV infected
patients with superior treatment options in the near future.5
We and others have advanced multiple classes of HCV DAAs
with complementary mechanisms of action with the goal of devel-
oping pegIFN-free therapies.6 We recently reported the discovery
of a novel series of anthranilic acid-based inhibitors that bind to
the allosteric Thumb Pocket 2 (TP-2) site of the HCV NS5B poly-
merase7,8 and complement our Thumb Pocket 1 (TP-1) inhibitor
deleobuvir (BI 207127) and protease inhibitor faldaprevir.9–11
O
O
O
OH
OH
O
NH
NH
O
O
S
O
S
O
1
2
IC50 0.22 µM
EC50 > 30 µM
IC50 0.16 µM
EC50 > 30 µM
Optimization efforts began by establishing SAR around the left
hand side (LHS) phenoxy group on the anthranilic acid scaffold
and thereby exploring interactions with this region of the binding
pocket (Fig. 1). Inhibitors were prepared using parallel synthesis in
⇑
Corresponding author.
E-mail address: timothy.stammers@boehringer-ingelheim.com (T.A. Stammers).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.102

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T. A. Stammers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6879–6885
5-substituted (4-methylphenylsulfonamides 1, 3–11) 4-substi-
tuted and (5-bromothien-2-ylsulfonamides 12-19) series are
shown Table 1. The majority of non-polar and polar substituents
around the phenoxy ring resulted in a loss of potency or a flat
SAR as demonstrated by analogues 3–7 and 13–17. The analogue
with a carboxylic acid at the para-phenoxy position 8 was a nota-
ble exception. Analysis of 8: NS5B co-structure highlighted a new
hydrogen bonding interaction between the LHS phenoxycarboxylic
acid and the backbone N–H of Leu497 as shown in Figure 1.14 In
the overlay with 1, the scaffold carboxylic acids of both inhibitors
maintain their interactions with the backbone N–H’s of Tyr477 and
Ser476 and the right hand side (RHS) 4-methylphenylsulfonamide
groups superimpose in a deep lipophilic pocket. However, signifi-
cant perturbations to the positioning and orientation of the anthra-
nilic acid and phenoxy rings of 8 occur in order to accommodate
the LHS H-bonding interaction. The improved enzymatic potency
of 8 did not translate into measurable cell culture activity, presum-
ably due to poor permeability associated with the presence of two
carboxylic acids. No improvement in potency was observed for the
isomeric phenoxy carboxylic acid analogue 18, suggesting that the
angle of approach did not allow the carboxylic acid of this isomer
to form a similar interaction with Leu497. The (ortho-trifluoro-
methyl)phenoxy analogue 9 proved to be an important discovery
from this exercise. Although no improvement in enzymatic
I482
Y477
S476
L419
L497
Figure 1. Overlay of NS5B co-structures of para-carboxylic acid 8 (white) with 1
(green).
the 5-substituted (e.g. 1) and 4-substituted (e.g. 2) anthranilic acid
series. Enzymatic (IC50, genotype 1b) and cell-based replicon
potencies (EC50, genotype 1b) of representative inhibitors in
Table 1
SAR around the LHS phenoxy group in the 5- and 4-substituted anthranilic acid sulfonamide series12,13
O
O
O
5
R
OH
OH
R
O
NH
S
NH
S
4
O
O
S
Br
O
O
R
GT1b-IC50
(
lM)
GT1b-EC50
>30
(
lM)
GT1b-IC50
0.61 0.2
(
lM)
GT1b-EC50
>25
(lM)
1
3
0.22 0.06
0.78 0.16
12
13
>30
>30
1.9 0.3
1.1 0.2
—
—
4
0.47 0.04
14
5
6
0.53 0.13
0.29 0.03
>30
30
15
16
1.5 0.4
2.9 0.4
—
—
O
N
H
O
HO
HO
7
0.33 0.11
>30
17
1.4 0.5
—
8
9
0.085 0.02
0.27 0.05
>30
18
19
2.2 0.6
3.0 0.6
—
O
7.3 2.5
>30
CF
3
10
11
0.37 0.21
0.21 0.05
7.0 1.7
8.6 4.9
CF
3
O

T. A. Stammers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6879–6885
6881
O
potency was observed relative to 1, a cell culture potency of
5.4 mM with no significant cytotoxicity (TC50 >100 mM) repre-
sented a noteworthy improvement for the anthranilic acid series.
Once again, the (ortho-trifluoromethyl)phenoxy analogue in the
4-substitued anthranilic acid series 19 failed to show such a bene-
fit. The lipophilic ortho-trifluoromethoxy analogue 10 also con-
ferred a similar benefit for cell culture potency (EC50 = 7.0 mM).
Among the non-aromatic replacements of the phenoxy group,
cyclopentyloxy analogue 11 was the only example that achieved
comparable potency (the binding of the lipophilic LHS groups of
9 and 11 is discussed in Fig. 3).
O
HO
O
OH
+ O
O
11
a, b
c, d
N
O
NH3Cl
20
21
Scheme 1. Reagents and conditions: (a) cyclopentanol (2.2 equiv), PPh3 (2.2 equiv),
DEAD (2.2 equiv), THF, 0.5 h, rt; (b) 10% Pd/C, H2, MeOH/EtOAc (1:1), 3 h, rt then
treatment with 1 N HCl in Et2O; (c) 4-Me-phenylsulfonyl chloride (1 equiv),
anhydrous pyridine, 80 °C, 1h; (d) NaOH (10 N aq, 10 equiv), DMSO, 50 °C, 2 h.
to improve cell culture potency. The 4-bromo-2-fluorophenylsulf-
onamide substituent7 present in the 5- (22) and 4-substituted
(27) anthranilic acid analogues served as references, both com-
pounds had comparable enzymatic potency but were inactive in
the cell-based replicon assay (Table 2). Methylation of the nitrogen
atom of the sulfonamide resulted in a complete loss of potency
(data not show), potentially due to unfavourable changes to the
conformation of the inhibitor. Replacement of the sulfonamide
with an N-alkylamide has been exemplified in the previously re-
ported thiophene carboxylic acid class of TP-2 inhibitors.15 Alkyl
substitution of the nitrogen atom was essential to lock the amide
in the s-cis bound-like conformation and retain activity. Increasing
steric volume of the nitrogen substituent from N-H ? N-Me ? N-
iPr led to a progressive improvement in potency (data not shown)
consistent with what was reported in the thiophene series.15 The
initial N-iPr amide inhibitors synthesized with the 4-bromo-2-fluo-
rophenyl substituent (23 and 28) were fivefold less potent than the
corresponding sulfonamides 22 and 27 and did not exhibit activity
in the replicon assay. The trans-4-methylcyclohexyl15 substituted
analogues 24 and 29 remained twofold less potent in the biochem-
ical assay compared to 22 and 27, however, weak inhibition was
measured in the cell culture assay. The SAR around the cyclohexyl
group was found to be highly restrictive. Removal of the trans-4-
methyl group resulted in a 30-fold loss of activity (data not
shown). Alternatives to the trans-4-methyl were generally less po-
tent, the best tolerated examples included the ethyl (25 and 30)
and trifluoromethyl (26 and 31) analogues. A comparison of the
binding mode of sulfonamide 22 with an N-iPr amide 24 is shown
in Figure 2. The key pharmacophoric interactions described in Fig-
ure 1 are well conserved as seen in the overlay.
The phenoxy substituted inhibitors in Table 1 were prepared
using a parallel synthesis approach described in our previous com-
munication.7 Cyclopentyloxy inhibitor 11 was prepared as shown
in Scheme 1. A Mitsunobu reaction was used to install the cyclo-
pentyl ring on 5-hydroxy-2-nitrobenzoic acid 20. Subsequently,
the nitro group was reduced to provide intermediate 21. Sulfonyla-
tion and saponification of 21 produced 11.
Simultaneous to our exploration of the phenoxy substituent,
modifications to the sulfonamide group were pursued in an effort
The synthesis of the sulfonamide inhibitors 22 and 27 was de-
scribed in our previous communication.7 The N-iPr amide inhibi-
tors from Table 2 were prepared from the phenoxy substituted
anthranilic acid methyl ester intermediates 32 and 337 as shown
in Scheme 2. Treatment with methoxypropene and sodium triacet-
oxyborohydride16 afforded N-iPr anilines 34 and 35. The synthesis
of 5-phenoxyanthranilic acid inhibitors 23–26 and 4-phenoxy-
anthranilic acid inhibitors 28–31 was completed by acylation and
saponification of the corresponding N-iPr-anilines 34 and 35.
Combinations of the best phenoxy substituents from Table 1 with
the trans-4-methylcyclohexyl-N-iPr carboxamide were explored
and our observations are summarized in Table 3. Gratifyingly, the
combination of the N-alkylamide linker with the 5-cyclopentoxy
Figure 2. Overlay of NS5B co-structures of sulfonamide 22 (white) and N-iPr-amide
24 (orange).
I482
A486
V485
group proved to be additive for cell culture potency (36, EC50
=
L489
L419
1.0 mM). Surprisingly, the corresponding 4-cyclopentyloxy
analogue 37 was inactive. The (ortho-trifluoromethyl)phenoxy
analogue also demonstrated an additive SAR resulting in the first
inhibitor in the series with sub-micromolar cell culture potency
(38, EC50 = 0.64 mM). The corresponding (ortho-trifluoromethyl)
phenoxy in the isomeric 4-substituted anthranilic acid series 39
failed to provide a comparable improvement in potency. The
trajectory of the phenoxy group from the 4-position of the anthra-
nilic acid did not appear to allow for the same beneficial interactions
observed for 36 and 38. All approaches to improve potency in the
4-substituted series failed and further optimization efforts focused
on the 5-substituted anthranilic acid series.
L497
M423
Figure 3. Overlay of NS5B co-structures comparing how the cyclopentyloxy 36
(white) ortho-trifluoromethylphenoxy 38 (magenta), and phenoxy 24 (blue) groups
fill the shallow lipophilic pocket on the LHS of TP-2.

6882
T. A. Stammers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6879–6885
Table 2
Replacement of the sulfonamide linker
O
O
O
5
OH
OH
O
R
4
R
R
GT1b-IC50
(l
M)
GT1b-EC50
>30
(l
M)
GT1b-IC50
(
lM)
GT1b-EC50
>30
(lM)
NH
O
S
22
0.34 0.18
1.64 0.1
27
28
0.31 0.08
O
Br
F
N
23
>10
1.4 0.3
>10
O
Br
F
N
24
25
0.67 0.26
5.0 1.3
29
30
0.74 0.30
11
6
O
N
1.3 0.8
25
16
3
7
4.3 1.6
>40
O
N
26
2.0 0.4
31
1.3 0.3
16 1.8
O
CF3
similar binding orientation to the phenoxy group of 24 though the
nature of the interactions reflects the difference between the aryl
and alkyl groups. In the case of 38, the trifluoromethyl effectively
fills the shallow pocket and the phenoxy group is projected away
from the protein surface towards solvent.
O
N
O
O
OH
O
O
O
O
O
c
d
NH3Cl
NH
R
O
Inhibitors from Table 3 were prepared as described in Scheme 3.
Esterification of 5-hydroxy-2-nitrobenzoic acid 20 followed by
benzylation of the phenolic oxygen provided intermediate 48.
Reduction of the nitro function was performed with iron powder
and the resulting aniline was alkylated using the reductive amina-
tion procedure described in Scheme 2. For the acylation of aniline
49, the use of freshly prepared trans-4-methylcyclohexylcarbonyl
chloride and anhydrous pyridine was necessary to ensure high
conversion on multigram scale. Removal of the benzyl protecting
group by hydrogenolysis provided phenol intermediate 50. A SNAr
reaction between 50 and either 2-fluoroacetophenone or 2-fluoro-
benzaldehyde provided diaryl ether intermediates 51 and 52. The
more reactive aldehyde 52 was converted to a difluoromethyl
group with Deoxofluor™ at room temperature but conversion of
the methylketone 53 to the difluoroethyl group required warming
the reaction mixture to 70 °C. Finally, saponification provided
inhibitors 44 and 45. Cyclopentyloxy inhibitor 36 was prepared
from intermediate 21 using protocols described in Scheme 2. The
isomeric cyclopentyloxy inhibitor 37 was prepared directly from
phenol intermediate 53 that can be accessed from the same
sequence of reactions that produced 50 starting from ethyl-4-
hydroxy-2-nitrobenzoate. Inhibitors 38–42 were prepared by SNAr
addition of corresponding phenols to either methyl-5-chloro-
2-nitrobenzoate or methyl-4-chloro-2-nitrobenzoate followed by
elaboration to the inhibitors as shown in Scheme 2. The
32 / 33
34 / 35
23-26 / 28-31
Scheme 2. Reagents and conditions: (a) NaH (1.2 equiv), MeI (3 equiv), DMF, rt, 1h;
(b) aq NaOH (10 N, 10 equiv), DMSO, rt, 12 h; (c) 2-methoxypropene (1.5 equiv),
Na(OAc)3BH (2 equiv), DCM, 2 h; (d) acyl chloride (2 equiv), anhydrous pyridine,
60 °C, 12 h then aq NaOH (5 N, 15 equiv), 50 °C, 4 h.
In the 5-substituted anthranilic acid series the isomeric meta-
trifluoromethyl analogue 40 failed to achieve the same cell culture
potency benefit as the ortho-analogue 38. Other hydrophobic ortho-
substitutions such as the trifluoromethoxy 41, bromo 42 and cyclo-
propyl 43 also failed to achieve a significant benefit over 24. The
closest structural analogue to 38, the (ortho-difluoroethyl)phenoxy
analogue 44 possessed comparable potency and the difluoromethyl
45, and tBu (EC50 = 0.84 mM, not shown in Table 3) analogues were
within twofold of 38. The incorporation of a nitrogen atom into the
(ortho-trifluoromethyl)phenoxy ring was also explored. An
improvement in potency was observed for both isomeric (ortho-tri-
fluoromethyl)pyridyloxy inhibitors 46 and 47 relative to the phen-
oxy analogue 38. The trifluoromethyl group of 46 was found to be
essential for activity, the unsubstituted pyrid-2-yloxy derivative
having an EC50 value of 16 mM (not shown in Table 3).
Figure 3 illuminates how the LHS substituents of 36, 38 and 24
occupy a shallow lipohilic pocket defined by residues L419, M423,
I482, V485, A486, L489, L497. The cyclopentyloxy group of 36 has a

T. A. Stammers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6879–6885
6883
Table 3
Phenoxy SAR with the trans-4-methylcyclohexyl-N-iPr carboxamide in the 5- and 4-substituted anthranilic acid series
O
O
O
5
R
OH
O
OH
O
R
N
O
N
4
R
GT1b-IC50
(l
M)
GT1b-EC50
5.0 1.3
(
l
M)
GT1b-IC50
(l
M)
GT1b-EC50
11 5.8
>50
(lM)
24
36
0.67 0.26
0.46 0.19
29
37
0.74 0.30
>40
1.0 0.05
0.63 0.3
38
40
0.19 0.12
0.47 0.06
39
1.3 0.8
4.5 0.9
CF3
6.3 0.9
CF3
41
42
0.74 0.2
0.45 0.1
2.8 1.1
4.9 0.2
CF3
O
Br
43
44
0.33 0.07
0.30 0.08
4.3 4.0
0.62 0.4
F
F
45
46
47
0.20 0.01
0.08 0.04
0.19 0.03
1.1 0.25
0.095 0.03
0.17 0.08
CHF2
N
CF3
CF3
N
(ortho-cyclopropyl)phenoxy analogue 43 was prepared from ortho-
bromophenoxy intermediate 54 in the presence of tricyclopropyl-
bismuth and (Ph3P)4Pd.17 The (ortho-trifluoromethyl)pyridyloxy
inhibitors 46 and 47 were prepared via a SNAr reaction between
50 and 2-fluoro-3-trifluoromethylpyridine or 4-fluoro-3-trifluo-
romethylpyridine with an in situ saponification.
Additional profiling of inhibitors 9, 38 and 46 is shown in Ta-
ble 4. All three compounds share the LHS ortho-trifluoromethyl
substitution. Inhibitor 38 replaces the 4-methyphenylsulfonamide
RHS of inhibitor 9 (Table 1) with a trans-4-methylcyclohexyl-N-
iPr carboxamide. Inhibitor 46 contains a pyrid-2-yloxy LHS ring
instead of the phenoxy ring of 38. A modest improvement in
enzymatic potency is observed across the three inhibitors but
the impact on replicon potency approaches 100-fold. A consider-
able improvement in Caco-2 permeability of the trans-4-meth-
ylcyclohexyl-N-iPr carboxamide is evident (38/46 vs 9). N-iPr
carboxamides 38 and 46 also have higher clogP values than sul-
fonamide 9. These properties may contribute to the improved cell
culture potency but other uninvestigated factors could also be
important (e.g. inhibitor binding kinetics, intracellular concentra-
tions). The incorporation of a nitrogen atom into the phenoxy ring
(46) improved potency in the enzymatic assay by 2.6- and 6.7-
fold in the replicon assay relative to 38. No experimental evi-
dence was generated to determine the origin of the benefit but
it could be speculated to be conformational or entropic through
solvation of the exposed face of the inhibitor (Fig. 3). The physi-
cochemical properties of the anthranilic acid inhibitors are heav-
ily influenced by the carboxylic acid as reflected by pH dependent
solubilities measured for 38 and 46. In addition, the measured
logD (pH 7.4) of 38 is 2.9, significantly lower than the clogP.
With measured pKa values of approximately 5.5, the anthranilic
acid TP-2 inhibitors are expected to be primarily in the ionized
state at pH 7.4. Both N-iPr-amide inhibitors were found to have
improved liver microsome stability (Human/Rat Liver Micro-
somes, HLM/RLM) over sulfonamide 9. ADME properties of
anthranilic acid inhibitors with the sulfonamide linker (Ref. 7
and unpublished results) suggest that the 4-methylphenylsulf-
onamide moiety is prone to phase 1 metabolism. Inhibitors 38
and 46 were orally bioavailable in rats in preliminary screening
experiments (data not shown) and the results warranted further

6884
T. A. Stammers et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6879–6885
O
O
O
para-carboxylic acid of 8 did not contribute to achieving cell cul-
ture potency in the work described herein. Nevertheless, elsewhere
we reported a series of non-acidic-TP-2 inhibitors that capitalized
on the Leu497 interaction for considerable gains in intrinsic and
cell culture replicon potency.18 A lack of productive SAR in the 4-
substituted anthranilic acid series (e.g. 39) allowed for prioritiza-
tion of the 5-substituted series. The incorporation of a nitrogen
atom into the (ortho-trifluoromethyl)phenoxy ring provided inhib-
itor 46, with enzymatic and cell culture potency less than 100 nM.
Further work on the anthranilic acid class of TP-2 inhibitors fo-
cused on improving the cell-based replicon potency (<10 nM)
and the pharmacokinetic properties with the goal of achieving a
preclinical profile consistent with an efficacious HCV direct acting
antiviral.19
HO
BnO
BnO
OH
O
O
a, b
c, d
e, f
NO2
NO2
N
H
20
48
49
R
O
O
N
O
N
HO
O
O
O
O
O
44, 45
g
h, i
50
51, 52
l
46, 47
Acknowledgments
O
Br
O
O
We thank the following colleagues from Boehringer Ingelheim
(Canada) Ltd that participated in generating the data presented
in this paper: Nathalie Dansereau, Carol Lawetz, Lisette Lagacée,
Ginette McKercher and Louise Thauvette for IC50 and EC50 determi-
nations. Josie De Marte, Edith Bellavance, Hélène Montpetit, Man-
on Rhéaume, Ma’an Amad and Jianmin Duan for ADME-PK support.
Sylvain Bordeleau, Colette Boucher and Michael Little for analytical
support.
O
O
O
43
j, k
HO
53
N
N
O
54
Scheme 3. Reagents and conditions: (a) SOCl2 (4 equiv in 3 portions), MeOH, reflux,
1 d; (b) benzylbromide (1.25 equiv), K2CO3 (4 equiv), acetone, rt, 17 h; (c) Fe0
(powder, 15 equiv), EtOH/AcOH (18:1), reflux, 5 h; (d) same a Scheme 2 (c); (e)
trans-4-methylcyclohexylcarbonyl chloride (2 equiv), anhydrous pyridine, DMAP
(0.1 equiv), 60 °C, 17 h; (f) 10% Pd/C, H2, EtOAc/MeOH (3:1), rt, 8 h; (g) 2-
fluoroacetophenone or 2-fluorobenzaldehyde (1.2 equiv), K2CO3 (2.5 equiv), DMSO,
100 °C, 0.5–24 h (h) R = H: deoxofluor (1.7 equiv), MeOH (cat.), DCM, 17 h, rt;
R = CH3: deoxofluor (8 equiv), MeOH (cat.), DCM, 24 h, 70 °C; (i) aq NaOH (2.5 N,
5 equiv), DMSO, rt, 1 h; (j) tricyclopropylbismuth (1.5 equiv), (PPh3)4Pd (0.1 equiv),
DMF, K2CO3 (2 equiv), 90 °C, 15 h; (j) aq NaOH (5 N, 10 equiv), DMSO/THF (1:1),
50 °C, 1 h; (l) 2-fluoro-3-trifluoromethylpyridine or 4-fluoro-3-trifluoromethylpyri-
dine (1.2 equiv), K2CO3 (2.5 equiv), DMSO then 5 N aq NaOH (10 equiv), 55 °C, 1 h.
References and notes
1. Marks, K. M.; Jacobson, I. Antiviral Ther. 2012, 17, 1119.
2. Hézode, C.; Dorival, C.; Zoulim, F.; Larrey, D. G.; Pol, S.; Cacoub, P.; Canva, V.;
Poynard, T.; Samuel, D.; Bourliere, M.; Alric, L.; Raabe, J.-J.; Zarski, J.-P. H.;
Riachi, G.; Bernard, P.-H.; de Ledinghen, V.; Loustaud-Ratti, V.; Metivier, S.;
Causse, X.; Marcellin, P.; Barthe, Y.; Fontaine, H.; Carrat, F.; Bronowicki, J.-P.
Hepatology 2012, 56, 217A.
3. Talal, A. H.; LaFleur, J.; Hoop, R.; Martin, P.; Jacobson, I.; Han, J.; Korner, E. J.
Aliment. Pharmacol. Ther. 2013, 37, 473.
4. (a) Shiffman, M. L.; Benhamou, Y. Liver Int. 2013, 33, 105; (b) McGowan, C. E.;
Fried, M. W. Liver Int. 2012, 32, 151; (c) Volk, M. L. J. Antimicrob. Chemother.
2010, 65, 1327.
Table 4
Comparison of the physicochemical and ADME properties of 9, 38 and 46
5. (a) Gane, E. Antiviral Ther. 2012, 17, 1210; (b) Pockros, P. J. Drugs 2012, 72, 1825;
(c) Lange, C. M.; Zeuzem, S. J. Hepatol. 2013, 58, 583.
9
38
46
MW
IC50 (mM)
451
0.27
7.3
5.6
—
—
3.6
12/—
463
0.20
0.64
7.1
2.9
<0.1 / 760
15
464
6. (a) Doyle, J. S.; Aspinall, E.; Liew, D.; Thompson, A. J.; Hellard, M. E. Br. J. Clin.
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S.; Jakalian, A.; Wernic, D.; Tsantrizos, Y. S.; Poupart, M.-A.; Bös, M.; McKercher,
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8. For recent review describing different classes of HCV NS5B inhibitors see: (a)
Beaulieu, P. L. Successful Strategies for the Discovery of Antiviral Drugs. In RSC
Drug Discovery Series; Desai, M. C., Meanwell, N. A., Eds.; RSC: Cambridge, 2013;
p 248. Vol. 32; (b) Sofia, M. J.; Chang, W.; Furman, P. A.; Mosley, R. T.; Ross, B. S.
J. Med. Chem. 2012, 55, 2481.
0.076
0.095
6.5
GT 1b EC50 (mM)
Calculated logP
Measured logD7.4
Sol. pH 2/6.8 (g/mL)a
Caco-2 (Â10À6 cm/s)b
HLM/RLM (t1/2, min.)c
—
<0.1 / >850
19
69 / 60
119 / 78
a
b
c
24 h solubility with amorphous solids in buffer using the shaking flask method.
Apical (pH 6) to basolateral (pH 7.4) permeability.
9. (a) Larrey, D.; Lohse, A. W.; de Ledinghen, V.; Trepo, C.; Gerlach, T.; Zarski, J.-P.;
Tran, A.; Mathurin, P.; Thimme, R.; Arastéh, K.; Trautwein, C.; Cerny, A.;
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W. O.; Steffgen, J. J. Hepatol. 2012, 57, 39; (b) Beaulieu, P. L.; Bös, M.; Cordingley,
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Shafran, S. D.; Pol, S.; Mauss, S.; Larrey, D.; Datsenko, Y.; Stern, J. O.; Kukolj, G.;
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Human and Rat Liver Microsomal (LM) stability at 2 lM starting concentration.
evaluation of the pharmacokinetic properties of this class of
inhibitors that will be reported in due course.
In conclusion, multiple observations emerged from this work
that contributed to establishing good cell culture potency for the
anthranilic acid-based TP-2 NS5B inhibitors. Replacement of the
arylsulfonamide RHS with the trans-4-methylcyclohexyl-N-iPr car-
boxamide in addition to the incorporation of the ortho-trifluoro-
methyl LHS substitution provided inhibitors with improved
permeability and cell culture activity (1 ? 38). Favorable interac-
tions with the shallow lipophilic pocket on the LHS of the binding
site were observed with the 5-(ortho-trifluoromethyl)phenoxy
substitution on the anthranilic acid scaffold (Fig. 3). While a mod-
est (38 vs 24) or no (9 vs 1) improvement in intrinsic activity was
observed, the interaction appears to contribute to a more pro-
nounced improvement in cell culture activity. The hydrogen bond-
ing interaction observed between the backbone N–H of Leu497 and
11. (a) Zeuzem, S.; Soriano, V.; Asselah, T.; Bronowicki, J.-P.; Lohse, A. W.;
Müllhaupt, B.; Schuchmann, M.; Bourtière, M.; Buti, M.; Roberts, S. K.; Gane, E.;
Stern, J. O.; Vinisko, R. A.; Kukolj, G.; Gallivan, J.-P.; Böcher, W. O.; Mensa, F. J. N.
Engl. J. Med. 2013, 369, 631; (b) Zeuzem, S.; Asselah, T.; Angus, P.; Zarski, J.-P.;

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6885
Larrey, D.; Müllhaupt, B.; Gane, E.; Schuchmann, M.; Lohse, A. W.; Pol, S.;
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D
21NS5B protein: McKercher, G.; Beaulieu, P. L.; Lamarre, D.; LaPlante, S.;
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a genotype 1b HCV replicon luciferase
reporter assay: (a) Vaillancourt, F. H.; Pilote, L.; Cartier, M.; Lippens, J.; Liuzzi,
M.; Bethell, R. C.; Cordingley, M. G.; Kukolj, G. Virology 2009, 387, 5; (b)
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(4JU3), 22 (4JU4), 24 (4JU7), 36 (4JU6) and 38 (4JJS) have been deposited in the
Protein Data Bank.
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